Machine for the field inoculation of corn with bacterial pathogens

ABSTRACT

An inoculation apparatus is disclosed whereby plants may be inoculated with a pathogen. The inoculation apparatus may includes a plurality of wound members and one or more pads. The pathogen is provided in an inoculum. In one example, the pressure of the inoculum is up to about 2 psi.

PRIORITY CLAIM

This application claims a priority based on provisional application61/001,502 which was filed in the U.S. Patent and Trademark Office onNov. 1, 2007, the entire disclosure of which is hereby incorporated byreference.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates generally to devices to inoculate plantlife with biological pathogens and in particular to inoculation devicesfor the inoculation of crops, such as corn, with bacterial pathogens.

Historically there has been a desire to screen Mycogen inbreeds andhybrids against the bacterial pathogens causing various diseases.Exemplary diseases include Stewart's Wilt and Leaf Blight (SWLB) andGoss's Wilt (GW).

The traditional method of an inoculation includes the use of two boardshinged together. The boards include a bed of nails embedded in aninoculum soaked sponge on the bottom of both boards. The bed of nailsserves to wound the plant and deliver the inoculum. The two boards areheld by an operator walking through the field and manually opened andclosed to wound each plant. This method is labor intensive and suffersfrom built-in inoculator and worker variation. Efforts have been made toproduce a machine that would simultaneously wound and inoculate a plant.

A prior art attempt was a recirculating solid stream spraying machinedeveloped for the inoculation of MDNV. The machine used a solid streamof inoculum to force viral particles into the tissue of the plantcausing a water soaked wound. The necessary pressure to cause thevisible water soaked wound was between 60 and 80 PSI. The inoculationdid not result in any visible symptoms on the plants and tests of theinoculum before and after showed the technique was killing most of thebacteria. It was believed that the violent decompression at the nozzlewas destroying the bacterial cells.

In a exemplary embodiment of the present disclosure, an inoculationssystem is provided. The inoculation system being for use with a vehicleto inoculate plants arranged in a row with a pathogen. The inoculationsystem comprising a frame supported by the vehicle and a feed systemwhich receives in series the plants arranged in the row. The feed systemalso provides a plurality of wounds in each of the received plants witha plurality of wound members and communicates the pathogen to theplurality of wounds. The inoculation system further comprises a drivesystem which moves the plurality of wounds members relative to thereceived plants.

In another exemplary embodiment of the present disclosure, aninoculations system for use with a vehicle to inoculate plants arrangedin a row with a pathogen is provided. The inoculation system comprisinga frame supported by the vehicle; means for capturing a plant of theplants arranged in the row; and means for wounding the plant captured bythe means for capturing, wherein the pathogen is introduced into thewounds of the plant captured by the means for capturing.

In a further exemplary embodiment of the present disclosure, a methodfor inoculating plants arranged in a row with a pathogen is provided.The method comprising the steps of driving a vehicle along the row, thevehicle transporting a plurality of moveable wound members and areservoir containing an inoculum containing the pathogen; capturing aplant of the row; wounding the plant with the plurality of wound memberscausing a plurality of wounds; and communicating the pathogen to theplurality of wounds.

In yet another exemplary embodiment of the present disclosure, a methodfor inoculating plants arranged in a row with a pathogen is provided.The method comprising the steps of driving a vehicle along the row at afirst speed, the vehicle transporting an inoculation system including aplurality of wound members and a reservoir containing an inoculumcontaining the pathogen; capturing a plant in the row between a firstmoveable member of the inoculation system positioned on a first side ofthe row and a second moveable member of the inoculation system on asecond side of the row, at least one of the first moveable member andthe second moveable member supporting the plurality of wound members;driving at least one of the first moveable member of the inoculationsystem and the second moveable member of the inoculation system at asecond speed, the second speed being based on the first speed of thevehicle; piercing the plant with at least a portion of the plurality ofwound members causing a plurality of wounds; and communicating thepathogen to the plurality of wounds.

In still a further exemplary embodiment of the present disclosure, anapparatus for inoculating plants in a field with a pathogen is provided.The plants being arranged in a plurality of rows. The apparatuscomprising a frame having a first frame portion and a second frameportion, the second frame portion being moveable relative to the firstframe portion; a plurality of ground engaging member coupled to thefirst frame portion; an engine operatively coupled to at least one ofthe plurality of ground engaging members to propel the apparatus throughthe movement of the at least one of the plurality of ground engagingmembers; a first moveable member positioned on a first side of a firstrow; and a second moveable member positioned on a second side of thefirst row. The plurality of ground engaging members being spaced to belocated between respective rows. The frame spanning at least one row.The first moveable member and the second moveable member cooperating tocapture a plant in the first row while the plant is wounded with aplurality of moveable wound members supported by least one of the firstmoveable member and the second moveable member. The apparatus furthercomprising a pathogen delivery system supported by the frame and a drivesystem coupled to at least one of the first moveable member and thesecond moveable member to move the plurality of moveable wounds membersrelative to the captured plants. The pathogen delivery system providingan inoculum containing the pathogen to the wounds of the plant caused bythe plurality of wound members.

Additional features and advantages of the present invention will becomeapparent to those skilled in the art upon consideration of the followingdetailed description of illustrative embodiments exemplifying the bestmode of carrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description of the drawings particularly refers to theaccompanying figures in which:

FIG. 1 is a representative view of a vehicle and an inoculation system;

FIG. 2 is a perspective view of an exemplary inoculation system;

FIG. 3 is a front view of a portion the inoculation system of FIG. 2;

FIG. 4 is a representative top view of the operation of the inoculationsystem of FIG. 2; and

FIG. 5 illustrates the infected lesions produced on a leaf subsequent towounding the leaf with the inoculation system of FIG. 2.

DETAILED DESCRIPTION OF THE DRAWINGS

The embodiments of the invention described herein are not intended to beexhaustive or to limit the invention to the precise forms disclosed.Rather, the embodiments selected for description have been chosen toenable one skilled in the art to practice the invention.

Referring to FIG. 1, an inoculation system 100 is represented.Inoculation system 100 wounds plants and provides an inoculum containingthe pathogen to the wounds of the plant. The pathogen is delivered at apressure to maintain the vitality of the pathogen.

Inoculation system 100 is configured for use with a vehicle 102. In oneembodiment, inoculation system 100 is supported by vehicle 102. In oneembodiment, inoculation system 100 is a separate unit coupled to vehicle102, such as a pull-behind unit.

Vehicle 102 includes a plurality of ground engaging members 104.Exemplary ground engaging members include wheels and tracks. Vehicle 102includes a vehicle frame 106 which supports, among other components, anengine 108 and a hydraulic system 110. As is known, engine 108 providespower to one or more of ground engaging members 104 to propel vehicle102 relative to the ground. Hydraulic system 110 provides a pressurizedhydraulic fluid, which may be used by hydraulic cylinders to adjust therelative placement or orientation of various components. Exemplaryvehicles include tractors, all-terrain vehicles, utility vehicles, andother suitable types of vehicles.

Inoculation system 100 includes a frame 120, which is coupled to vehicleframe 106. Inoculation system 100 further includes a feed system 122,which is positioned to align with a row 130 (see FIG. 4) of plants 132and to provide the wounds on each plant. Referring to FIG. 4, plants 132are arranged in a plurality of rows 130. In general, feed system 122 issized to interact with a given row 130 of plants without causing damageto adjacent rows 130 of plants 132.

A height adjustment system 124 is provided to adjust the relative heightof feed system 122 relative to the ground. Exemplary height adjustmentsystem includes one or more hydraulic cylinders coupled to the hydraulicsystem 110 of vehicle 102. The one or more hydraulic cylinderscooperative with frame 120 to adjust the height of feed system 122. Inone embodiment, height adjustment system 124 also adjusts the lateralposition of feed system 122.

Inoculation system 100 further includes a pathogen delivery system 126,which provides the biological pathogens to feed system 122 forapplication to the plants. In one embodiment, the biological pathogensare provided in a liquid inoculum that is stored in a reservoir of thepathogen delivery system 126. Exemplary biological pathogens includeErwinia stewartii and Clavibacter michaganensis subsp. nebraskensis.

Inoculation system 100 further includes a drive system 128. Drive system128 controls the operation speed of feed system 122. In one embodiment,drive system 128 operates feed system 122 at a first speed generallyequal to a ground speed of vehicle 102. In one embodiment, drive system128 operates feed system 122 at a speed slightly higher than the groundspeed of vehicle 102.

Inoculation system 100 may be a single row device in that it provideswounds on plants 132 of a single row as vehicle 102 is propelled along arow. An exemplary single row device is shown in FIGS. 2-4. Inoculationsystem may be a multi-row device in that it provides wounds on plants132 of multiple rows 130 as vehicle 102 is propelled along a row. Amulti-row device may consist of multiple single row devices coupledtogether, such as the exemplary single row device shown in FIGS. 2-4.

Referring to FIGS. 2-4, an exemplary inoculation system 200 is shown.Referring to FIG. 3, a front view of inoculation system 200 is shown.Inoculation system 200 includes a frame 202 and a feed system 204. Frame202 is coupled to vehicle 102, such as a tractor. In one embodiment,frame 202 is coupled to a tractor between a front ground engaging member104 of the tractor and a rear ground engaging member 104 of the tractorand to one side of the tractor. The frame 202 positions feed system 204to align feed system 204 with a row 130 in a field. In one embodiment, aheight adjustment system (not shown) is provided to raise and lower feedsystem 204 to vertically align feeds system 204 with the desiredportions of plants 132, such as leaves 134 (see FIG. 5). In oneembodiment, at least a portion of frame 202 is moveable by the heightadjustment system to align feed system 204. An exemplary heightadjustment system is one or more hydraulic cylinders which areoperatively coupled to hydraulic system 110 of vehicle 102 and move aportion of frame 202 relative to the ground.

Referring to FIG. 4, feed system 204 includes a first rotating member206, illustratively a cylinder, and a second rotating drum 208,illustratively a cylinder. Rotating members 206 and 208 are supported byshafts 210 and 212, respectively. A drive system 214 (see FIG. 3) drivesthe rotation of at least one of shafts 210 and 212 thereby causing therotation of rotating members 206 and 208. Rotating member 206 rotates indirection 216. Rotating member 208 rotates in direction 218.

Rotating member 206 and rotating member 208 support pads 220 and 222,respectively. Pad 222 is made of a compressible material which alsoholds an inoculum. As pad 222 is compressible portions of the inoculumare released from pad 222. In the illustrated embodiment, both pad 220and pad 222 are made of compressible materials and/or materials whichhold an inoculum. Exemplary compressible materials which hold aninoculum include foams, sponges, and other suitable materials. Pads 220and 222 are shown surrounding rotating members 206 and 208,respectively. In one embodiment, pads 220 and 220 may be localized to aparticular region of rotating members 206 and 208, respectively.

Pads 220 and 222, as shown in FIG. 4, are compressed in a region 230between rotating member 206 and rotating member 208. Region 230generally corresponds to a track which receives plants 132 as vehicle102 moves in direction 232. Pads 220 and 222 cooperate to capture aplant 132. In one embodiment, as described herein, the rotation speed ofrotating members 206 and 208 is controlled to be generally equal orslightly greater than a speed of vehicle 102 in direction 232. Thisprevents the plant 132 from being uprooted during capture. Pads 220 and222 while capturing plant 132 do not break plant 132 are due to thecompressive-nature of pads 220 and 222.

Rotating member 206 further supports a plurality of wounds members 234.Wound members 234 are generally sharp instruments which pierce orotherwise cut plant 132. As shown in FIG. 4, eight groupings of radiallyextending wound members 234 are provided generally equally spaced aboutrotating member 208. As shown in FIG. 3, each grouping includes aplurality of vertically offset wound members 234. Although eightgroupings are illustrated more or less groupings may be used. Further,wound members 234 may be spaced about rotating member 208 in any desiredpattern, whether in groupings or not. In addition, in one embodiment,both rotating members 206 and 208 support wound members. Exemplarywounds members include nails, blades, and other sharp instruments.

In one embodiment, wound members 234 are arranged in nine horizontalrows. Each row extends about a circumference of rotating member 208 witha spacing of about one half inch between adjacent wound members 234 inthe row. Further, the spacing between adjacent rows of wound members 234is about one half inch. The respective wound members 234 in each row arenot aligned with the wound members in the adjacent rows. In one example,with the spacing between the wound members 234 in a given row beingabout one half inch, the wound members 234 in the adjacent rows areoffset by about one quarter of an inch. As such, the wound members 234in the two rows on either side of an intermediate row are generallyvertically aligned and offset from the wound members 234 in theintermediate row by about one quarter of an inch.

As shown in FIG. 4, pad 222 in its uncompressed state, outside of region230, covers wound members 234. When pad 222 is in its compressed state,generally within region 230, wound members 234 extend beyond pad 222.Further, wound members 234 extend into pad 220.

In one embodiment, wound members 234 are nails and rotating member 208is a hollow cylinder. The nails are located from the inside of thecylinder and extend radially outward into pad 222. In one example, thenails are 1 inch nails which extend approximately 0.75 inches from anouter surface of the cylinder and pad 222 is a foam layer having athickness of about 1 inch. As such, the nails are covered byapproximately 0.25 inches of foam when pad 222 is in an uncompressedstate and extend beyond the foam by approximately 0.25 inches when pad222 is in a compressed state.

Turning to FIG. 3, feed system 204 is driven by drive system 214. Drivesystem 214 includes a drive wheel 250 which engages the ground androtates as vehicle 102 moves in direction 232. Drive wheel 250 iscoupled to an input pulley 252 through a shaft 254. Input pulley 252 iscoupled to an output pulley 256 through a drive belt 258. Output pulley256 is coupled to a first gear 260 through a shaft 262. First gear 260is engaged with a second gear 264 which is coupled to shaft 210. Assecond gear 264 is rotated by first gear 260 rotating member 208 isrotated. Due to the contact between rotating members 206 and 208 inregion 230, the rotation of rotating member 208 in direction 218 resultsin the rotation of rotating member 206 in direction 220.

In one embodiment, drive system 214 drives the rotation of rotatingmembers 206 and 208 at a speed generally equal to the ground speed ofvehicle 102. In one embodiment, drive system 214 drives the rotation ofrotating members 206 and 208 at a speed slightly higher than the groundspeed of vehicle 102. This permits feed system 204 to pull the plants132 through quicker and release them prior to the plant 132 being pulledout by the roots.

In one example, rotating members are about 4.5 inches in diameter. Pad220 and pad 222 are each about 1.0 inches thick, but are compressed toabout 0.5 inches thick in region 230. This results in an effectivediameter of rotating members 206 and 208 being about 5.5 inches. Drivewheel 250 has a diameter of 13 inches. As such, the ratio of theeffective diameter of rotating members 206 and 208 to the diameter ofdrive wheel 250 is about 0.432. The relative sizes of input pulley 252and output pulley 256 are selected to compensate for the non-unity ratioof the effective diameter of rotating members 206 and 208 to thediameter of drive wheel 250. A diameter of input pulley 252 is 6 inches.To provide an exact speed match between drive wheel 250 and rotatingmembers 206 and 208, the diameter of output pulley 256 should be 2.54. A2.5 inch diameter output pulley 256 was selected. This results in thespeed of rotating members 206 and 208 being slightly higher than thespeed of drive wheel 250 which is equal to the ground speed of vehicle102.

In one embodiment, vehicle 102 is a tractor having two spaced apartfront wheels 104 and drive wheel 250 is positioned in line with one ofthe front wheels of the tractor. The spacing of the front wheels isselected to permit the tractor to travel through the field with eachfront wheel located between two adjacent rows 130. In one example,region 230 and hence the row aligned with feed system 204 is offset froma centerline of drive wheel 250 by about 15 inches.

As mentioned herein, at least portions of frame 202 may raised orlowered with a height adjustment system, such as hydraulic cylinders. Atensioning member 270 couples drive wheel 250 to frame 202. In thismanner inoculation system 200 may be raised to permit travel on a roadsurface without drive wheel 250 engaging the road surface. Exemplarytensioning members include the illustrated chain.

Referring to FIG. 2, an exemplary pathogen delivery system 300 is shown.Pathogen delivery system 300 communicates an inoculum containing thepathogen from a reservoir 302 to one or both of pads 220 and 222.Pathogen delivery system 300 maintains the integrity of the pathogen toensure that it is able to interact with the plants 132 to which it isultimately communicated. Pathogen delivery system 300 maintains thepressure experienced by the inoculum at up to about 2 pounds per squareinch (psi).

Reservoir 302 is supplied with a quantity of inoculum. The location ofreservoir 302 is selected such that gravity will cause the inoculum toflow through a fluid conduit 304 towards a nozzle 306 positionedproximate to rotating members 206 and 208. Nozzle 306 has an associatedadjustable regulator 308 which sets the pressure that the fluid in fluidconduit 304 should be at or above to exit nozzle 306.

In addition to gravity, a pump 310 is in fluid communication with a topportion of reservoir 302. Pump 310 is an air pump which provides avolume of pressurized air above the inoculum in reservoir 302. In oneexample, pump 310 is a twelve volt tire pump. Reservoir 302 alsoincludes a 0-5 psi relief valve or pressure regulator 314 to keep pump310 from over-pressurizing the volume of air above the inoculum. In oneexample, the relief valve 314 is set to about 2 psi, such that thevolume of air may be pressurized up to a pressure of about 2 psi. In oneembodiment, a pressure gauge 316 is provided to provide a visualindicator to an operator of the pressure in fluid conduit 304.

The pathogen delivery system may be setup in the following manner. Theinoculum is placed in reservoir 302. Regulator 308 associated withnozzle 306 is set to about 0 psi. This resulted in a drip from thenozzle due to gravity. Pump 310 is activated and relief valve 314 is setto about 0 psi. Relief valve is adjusted until a steady drip/spray isexiting nozzle 306. In one embodiment, the pressure setting of reliefvalve 314, and hence the pressure of the volume of air in reservoir 302,corresponding to a steady drip/spray exiting from nozzle 306 is about 2psi. Nozzle 306 is positioned to direct the inoculum onto pad 222 and/orpad 220.

Inoculation system 200 may be used in the following manner. The pathogendelivery system 300 is setup to provide a steady drip/spray of inoculumfrom nozzle 306. This communicates the inoculum to pad 222 and/or pad220. For purposes of discussion, the inoculum is discussed in relationwith pad 222. Pad 222 absorbs or holds the inoculum.

Vehicle 102 is positioned such that region 230 of feed system 204 isgenerally aligned with a given row 130 of plants. Vehicle 102 isadvanced in direction 232 causing drive wheel 250 of drive system 214 torotate. The rotation of drive wheel 250 results in the rotation ofrotating members 206 and 208 in directions 216 and 218, respectively.

As a plant 132 of row 130 is encountered, it is captured between pad 220and pad 222. The advancement of plant 132 within region 230 compressespad 220 and pad 222 to bring a portion of wound members 234 into contactwith leaves 134 of plant 132. Wound members 234 pierce leaves 134resulting in wounds in leaves 134. As wound members 234 are piercingleaves 134, the inoculum being held by pad 222 and/or pad 220 is forcedout of the pad due to the compression of the pad. Some of the inoculumclings to the wound members 234.

As plant 132 further advances within region 230, the wound members 234are retracted relative to the leaves 134 due to the further rotation ofrotating member 208. At the same time pad 222 is expanding back towardsits uncompressed state. As pad 222 expands it wipes the inoculum offwound members 234. It is believed that this causes the inoculum to beforced into the edges of the wounds created by the wound members 234(which may still be partially in the wound); thereby providing a safeharbor for the organism to initiate an infection. The result of thisprocess is the development of lesions 138 (see FIG. 5) on the leaves 134of plants 132. The location of lesions 138 correspond to the locationsof the wounds produced by wound members 234.

Other attempts used a wire brush to create wounds in the plants and thepathogen delivery system 300. However, with this setup lesions 138 werenot adequately caused. It is believed that system 200 is able to causelesions 138 because it forces the inoculum into the edges of the woundscreated by the wound members 234.

Although the invention has been described in detail with reference tocertain preferred embodiments, variations and modifications exist withinthe spirit and scope of the invention as described and defined in thefollowing claims.

1. An inoculation system for use with a vehicle to inoculate plantsarranged in a row, the inoculation system comprising: a frame supportedby the vehicle; a feed system which receives in series the plantsarranged in the row, provides a plurality of wounds in each of thereceived plants with a plurality of wound members, and communicates apathogen to the plurality of wounds; and a drive system which moves theplurality of wound members relative to the received plants, the drivesystem including a drive wheel which engages the ground and rotates asvehicle moves, the drive wheel being operatively coupled to theplurality of wound members to power movement of the plurality of woundmembers.
 2. The inoculation system of claim 1, wherein the feed systemincludes a first rotating member and a second rotating member, theplants arranged in the row being received between the first rotatingmember and the second rotating member.
 3. The inoculation system ofclaim 2, wherein at least one of the first rotating member and thesecond rotating member support a pad which holds the pathogen andcommunicates the pathogen to the plurality of wounds provided in thereceived plants.
 4. The inoculation system of claim 3, wherein the padis compressed in a region between the first rotating member and thesecond rotating member and the pathogen is forced out of the pad and tothe plurality of wounds provided in the received plants due to thecompression of the pad.
 5. The inoculation system of claim 4, whereinthe pad is a foam layer.
 6. The inoculation system of claim 4, furthercomprising a pathogen delivery system which communicates the pathogen tothe pad.
 7. The inoculation system of claim 6, wherein the pathogendelivery system comprises a fluid reservoir holding an inoculumcontaining the pathogen; a fluid conduit in fluid communication with thefluid reservoir; a nozzle in fluid communication with the fluid conduit,the nozzle communicating the inoculum onto the pad; a pressure regulatorwhich controls a pressure of the inoculum provided to the nozzle; and apump which provides a positive pressure on the inoculum prior to beingexpelled from the nozzle.
 8. The inoculation system of claim 7, whereinthe pump is in fluid communication with the reservoir and increases thepositive pressure on the inoculum within the reservoir.
 9. Theinoculation system of claim 8, wherein the pump provides a pressurizedvolume of air in the reservoir above the inoculum and the pressureregulator is a relief valve in fluid communication with the pressurizedvolume of air.
 10. The inoculation system of claim 9, wherein thepressure regulator keeps the pressurized volume of air with a pressureof up to about 2 psi.
 11. The inoculation system of claim 2, wherein atleast one of the first rotating member and the second rotating membersupport the plurality of wound members, the drive system rotating the atleast one of the first rotating member and the second rotating member tomove the plurality of wound members relative to the received plants. 12.The inoculation system of claim 11, wherein the plurality of woundmembers are spaced apart radially extending pointed instruments.
 13. Theinoculation system of claim 12, wherein the pointed instruments arenails.
 14. The inoculation system of claim 12, wherein at least one ofthe first rotating member and the second rotating member support a padwhich carries the pathogen and communicates the pathogen to theplurality of wounds provided in the received plants by the plurality ofspaced apart radially extending pointed instruments, wherein the pad iscompressed in a region between the first rotating member and the secondrotating member and the pathogen is forced out of the pad and to theplurality of wounds provided in the received plants due to a compressionof the pad.
 15. The inoculation system of claim 14, wherein the padcoats the plurality of spaced apart radially extending pointedinstruments with the pathogen.
 16. The inoculation system of claim 1,wherein the drive system moves the plurality of wound members at a speedapproximate to a ground speed of the vehicle.
 17. An inoculation systemfor use with a vehicle to inoculate plants arranged in a row, theinoculation system comprising: a frame supported by the vehicle; a feedsystem which receives in series the plants arranged in the row between afirst rotating member of the feed system and a second rotating member ofthe feed system, provides a plurality of wounds in each of the receivedplants with a plurality of wound members, and communicates a pathogen tothe plurality of wounds, the plurality of wound members are spaced apartradially extending pointed instruments; and a drive system which movesthe plurality of wound members relative to the received plants, whereinat least one of the first rotating member and the second rotating membersupport the plurality of wound members, the drive system rotating the atleast one of the first rotating member and the second rotating member tomove the plurality of wound members relative to the received plants andwherein at least one of the first rotating member and the secondrotating member support a pad which carries the pathogen andcommunicates the pathogen to the plurality of wounds provided in thereceived plants by the plurality of spaced apart radially extendingpointed instruments, wherein the pad is compressed in a region betweenthe first rotating member and the second rotating member and thepathogen is forced out of the pad and to the plurality of woundsprovided in the received plants due to a compression of the pad, whereinwhile a radially extending pointed instrument is in a region between thefirst rotating member and the second rotating member a point of theradially extending pointed instrument extends out of the pad and whilethe radially extending pointed instrument is outside of the region thepoint of the radially extending pointed instrument is covered by thepad.